Transgender men and transmasculine youth are at high risk for acquiring HIV. Growing research on transgender men demonstrates increased HIV risk and burden compared with the general US population. Despite biomedical advancements in HIV prevention, there remains a dearth of evidence-based, sexual health HIV prevention interventions for young transgender men. MyPEEPS (Male Youth Pursuing Empowerment, Education, and Prevention around Sexuality) Mobile is a web-based app that builds on extensive formative community-informed work to develop an evidence-based HIV prevention intervention. Our study team developed and tested the MyPEEPS Mobile intervention for 13- to 18-year-old cisgender young men in a national randomized controlled trial, which demonstrated efficacy to reduce sexual risk in the short term-at 3-month follow-up. Trans men and transmasculine youth resonated with basic HIV educational information and sexual scenarios of the original MyPEEPS app for cisgender men, but recognized the app's lack of transmasculine specificity. The purpose of this study is to detail the user-centered design methods to adapt, improve the user interface, and enhance the usability of the MyPEEPS Mobile app for young transgender men and transmasculine youth. The MyPEEPS Mobile app for young transgender men was adapted through a user-centered design approach, which included an iterative review of the adapted prototype by expert advisors and a youth advisory board. The app was then evaluated through a rigorous usability evaluation. MyPEEPS Mobile is among the first mobile health interventions developed to meet the specific needs of young transgender men and transmasculine youth to reduce HIV risk behaviors. While many of the activities in the original MyPEEPS Mobile were rigorously developed and tested, there was a need to adapt our intervention to meet the specific needs and risk factors among young transgender men and transmasculine youth. The findings from this study describe the adaptation of these activities through feedback from a youth advisory board and expert advisors. Following adaptation of the content, the app underwent a rigorous usability assessment through an evaluation with experts in human-computer interaction (n=5) and targeted end users (n=20). Usability and adaptation findings demonstrate that the MyPEEPS Mobile app is highly usable and perceived as potentially useful for targeting HIV risk behaviors in young transgender men and transmasculine youth.